The molecular signals that allow progenitors to traffic to the thymus from the blood are very poorly understood. Work ongoing in our laboratory indicates that different hematopoietic progenitors differ in their ability to settle within the thymus from the blood. Hence downstream multipotent progenitors (MPPs) but not hematopoietic stem cells (HSCs) both circulate in blood, but only MPPs are able to settle within the thymus if placed in the blood. We have further fractionated MPPs, and find that only the early lymphoid progenitor subset (ELP) of MPPs, that expresses the lymphoid-specific genes RAG1 and RAG2, appears able to efficiently settle within the thymus, whereas other MPP subsets do not appear able to migrate into the thymus. This grant application is focused on understanding the molecular basis of the selective ability of ELPs to settle within the thymus. Our data thus far suggest that expression of the chemokine receptor CCR9 is regulated between ELPs that can settle within the thymus and RAG-negative MPPs and HSCs that cannot. Our data further indicate that regulated expression of CCR9 is important for the ability of ELPs to physiologically settle within the thymus. However, our preliminary data also indicate that additional molecules must be involved in the ability of ELPs to undergo regulated thymic settling. In Aim 1, we propose to identify molecules in addition to CCR9 whose expression is regulated between HSCs and MPPs. In Aim 2, we propose to perform genetic gain-of-function experiments to determine whether CCR9 and other identified molecules are sufficient to confer thymic homing on progenitors such as HSCs that normally fail to undertake this migration; and to perform loss of function experiments to determine whether such molecules physiologically play a role in the settling of circulating hematopoietic progenitors within the thymus. Together, these studies will identify the signals critically important in the physiological homing of progenitors to the thymus, and may provide new avenues for therapeutic interventions in circumstances where thymic reconstitution needs to be improved, such as improving intrathymic T cell reconstitution following bone marrow transplantation, and in aging. KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Avinash Bhandoola AVINASH University of PA P.I. OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Paul E. Love National Institutes of Health, MD Collaborator Wolfgang Weninger Wistar Institute, Philadelphia, PA Collaborator Donald A. Baldwin University of Pennsylvania, PA Collaborator The signals that direct the settling of hematopoietic progenitors within the thymus of adult mice are unknown. We propose to identify homing molecules expressed by hematopoietic progenitors that are able to settle within the thymus, and to determine the role of these molecules in the process of thymic settling. These experiments will allow a molecular definition of thymus-settling progenitors, and may enable therapeutic enhancement of T lineage reconstitution in clinical settings such as aging and bone marrow transplantation. [unreadable] [unreadable] [unreadable]